Root system exhibits hierarchical traits in physiology and exudation, and responds variably to environmental changes. However, it remains unclear if these phenomena result in corresponding alterations in the microbial characteristics of rhizosphere soil and diverse responses to nitrogen (N) deposition. This study evaluated microbial biomass, gas flow, and enzyme activity in rhizosphere soil of three root diameter classes (R1, <0.5 mm; R2, 0.5–1.0 mm; R3, 1.0–2.0 mm) of P. tabuliformis, and their responses to N application (0, 3, 6, 9 g N m−2 y−1, corresponding to CN, LN, MN, and HN) in a P. tabuliformis forest. Rhizosphere soil of R1 exhibited significantly higher microbial biomass N (MBN) content, enzyme activities, CO2 and N2O flow rates than those of R2 and R3 (P < 0.05). Soil microbial biomass carbon (MBC) and phosphorus (MBP) contents, N2O and CH4 flow rates significantly increased with N applications. While soil MBN content increased with the LN treatment and decreased with the HN treatment, the largest value was observed in the LN treatment (84.89 mg kg−1). N application promoted Alkaline phosphatase (ALP) and Alanine transaminase (ALT) activities in R1 rhizosphere soil while decreasing their activities in R2 and R3 rhizosphere soils. Moreover, β-glucosidase (BG), N-acetyl-β-Glucosaminidase (NAG), β-xylosidase (XYL), and β-D-cellulosidase (CBH) activities in R1 rhizosphere soil were inhibited, while their activities in R2 and R3 rhizosphere soils were promoted by N application. N application directly promoted microbial biomass then inhibited enzyme activity. Besides, nutrient content of rhizosphere soil decreased with root diameters, which indirectly reducing enzyme activity and microbial biomass. N application directly affected soil CH4 flow rate, while N2O flow rate was indirectly promoted by N application through increasing microbial biomass. CO2 and CH4 flow rates were inhibited by N application through decreasing enzyme activity. Root diameter slowed down the CO2 and CH4 flow rates by reducing soil nutrient content and enzyme activity. In conclusion, N deposition has varied effects on microbial characteristics in rhizosphere soil of different diameter roots, which are relevant to the corresponding changes in root system and soil properties.